Pseudomonas aeruginosa, a common inhabitant of water and soil, is also responsible for a range of serious human infections including the chronic respiratory disease of patients with cystic fibrosis (CF). The goal of this proposal is to apply molecular tools to define genetic variations within the genomes of various P. aeruginosa isolates. The project will test the hypothesis that unlike most strains of P. aeruginosa, the flexible gene pool of strains that are capable of colonizing patients with CF, consists of a specific repertoire of genes encoded within horizontally-acquired genes, often present in genomic islands. Moreover, during the chronic phase of infection these strains accumulate pathoadaptive mutations in the P. aeruginosa genome, which allow the bacteria to persist for many years in the respiratory tract. First, using DNA microarrays, analyzing variable segments of DNA by PCR, applying a targeted DNA capture method, and a differential hybridization strategy, we will identify genes in environmental and clinical strains which are not part of the core gene set and therefore may reside in genomic islands. A DNA microarray will be constructed which consist entirely of the genes from this flexible gene pool and it will be used to further analyze the changes in the genomes of a large set of CF isolates, including established lineages of early and late isolates from individual CF patients. The array will be also used to generate a transcriptome of in vivo expressed genes within the CF-specific islands, which will be used to guide the subsequent prioritization of relevant genes. In the second aim, those genomic island-containing genes that are (i) present in great majority of CF isolates, and (ii) expressed in animal models of infection, will be deleted or individual genes will be mutagenized. The effect of these mutations on the virulence of P. aeruginosa will be assessed in two animal models of respiratory infection. In the third aim, the appearance of point mutations in the genome will be also monitored in the clones of P. aeruginosa isolated over several years from individual CF patients. These mutations may be a required for P. aeruginosa to cause a chronic, long-lasting infection in the CF respiratory tract. This hypothesis will be tested in a rat chronic infection model, where accumulation of mutations will be compared to those seen in human infections. The results of the studies proposed in this application should provide new insights into the evolution of a highly successful opportunistic pathogen and shed light on some unexpected virulence mechanisms that function during chronic respiratory tract infections. Moreover, findings from this work should greatly supplement the currently available genomic resources for the research community, particularly those who work with different P. aeruginosa isolates. The availability of a "virtual genome", based on complete or partially-completed genome sequences as well as sequences of horizontally-acquired islands, should result in an improved understanding of the interplay between strain specific and core virulence determinants. [unreadable] [unreadable]